PROJECT SUMMARY Invasive fungal infections are an increasing public health threat and current antifungal therapy is extremely difficult due to the emergence of multi-drug or totally resistant strains as well as new pathogenic species. Because the biology of these organisms is unique compared to other common microbial pathogens, the arsenal of drugs to treat invasive fungal infections frequently exhibits limited efficacy and toxicity. In this program, we propose to conduct a series of screens using a compound library that has shown great success in providing lead compounds and preclinical candidates for oncology and metabolic diseases, bacterial infections (pan-Gram negative bacteria, Clostridium difficile-specific, and Mycobacterium tuberculosis), and parasitic diseases (Trypanosoma cruzi and Plasmodium falciparum). This library, assembled using Diversity-Oriented Synthesis (DOS) principles, contains a unique collection of chemical structures that possess enhanced topological and stereochemical diversity, resulting in three-dimensional characteristics not commonly represented in standard commercial libraries for high-throughput screening. We have assembled a unique multi-disciplinary team with complementary expertise in screening, chemical synthesis, fungal pathogenesis, in vitro and in vivo fungal models of disease, and the clinical treatment of invasive fungal infections. Using a miniaturized primary screen against four different clinically-relevant fungal pathogens (Candida albicans, Candida glabrata, Candida auris, Cryptococcus neoformans) and subsequently profiling hits against a wide range of yeasts, invasive molds (including Aspergillus fumigatus), and drug-resistant isolates will allow rapid and cost-effective generation of abundant activity profile data to prioritize compound series demonstrating either species-specific or broad-spectrum antifungal activities via novel mechanisms of action. Molecular targets will be identified via chemical genomic approaches enabling integration of biochemical and structural biology approaches in the compound optimization workflow. Due to our extensive experience in advancing DOS library hits to validated probes and preclinical candidates, we are able to rapidly optimize compound series for potency, physicochemical properties, and metabolic stability to deliver leads suitable for profiling in in vivo infection animal models. Our goal for this 5-year project is to identify, optimize, and validate 1 to 2 novel antifungal therapeutic leads for clinical development.